Method of rational-based drug design using osteocalcin

Abstract

The invention relates to a method of identifying a compound that affects osteocalcin activity, comprising obtaining a 3D structure of osteocalcin or a fragment thereof, designing a compound to interact with, or mimic, the 3D structure of osteocalcin or fragment thereof, obtaining the compound, and determining whether the compound affects osteocalcin activity.

Description

CROSS-REFERENCE TO RELATED APPLICATION[0001]This application is a continuation of U.S. application Ser. No. 10 / 972,690, filed on Oct. 25, 2004 which claims priority from U.S. provisional patent application No. 60 / 562,237, filed on Apr. 15, 2004 and Canadian application no. 2,446,527 filed on Oct. 23, 2003, both of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION[0002]The invention relates to the crystalline form of osteocalcin (“OC”). The invention also relates to methods of using the three-dimensional structure of osteocalcin to design and identify candidate compounds that will activate or inhibit osteocalcin activity. The invention also includes compounds identified using the methods of the invention. The invention also includes osteocalcin derivatives that act to inhibit osteocalcin-hydroxyapatite binding. Furthermore, the invention relates to the use of these compounds / derivatives in the treatment of diseases or degenerative conditions resultin...

AI Technical Summary

Benefits of technology

[0014]Now that the three-dimensional structure of the osteocalcin crystal has been determined, an inhibitor / modulator of osteocalcin-hydroxyapatite binding can be identified through the use of rational drug design by computer modeling with a docking program. This procedure can include computer fitting of potential inhibitors to the osteocalcin-hydroxyapatite binding to ascertain how well the shape and the chemical structure of the potential modulator will bind to hydroxyapatite to compete out osteocalcin. Computer programs can also be employed to estimate the attraction, repulsion, and steric hindrance of the subunits with a modulator / inhibitor. A particular advantage is that selective inhibitors can be identified by comparing the potential inhibitor to the 3D structure of osteocalcin.

[0024]In another embodiment, only the features on the HA binding surface are altered. This will direct the osteocalcin derivatives to locations other than bone and allow it to compete with the bone-bound osteocalcin in interacting with cellular protein and reduce the recruitment of cell to bone. Such osteocalcin derivative may include, but are not limited toi) de-carboxylation of Glas by chemical means or by expressing osteocalcin in host cells that cannot synthesize GLA.ii) Mutation of residues on the surface, as can be deduced from the 3-D structure (including but not limiting to Gla-17, Gla-21, Gla-24, Asp-30, Asp-34), that can cause steric clash with the hydroxyapatite surface and therefore prevent the OC mutant from interacting with the HA surface.

Problems solved by technology

Furthermore, bony metastases of cancers, including breast, prostate and lung cancers, cause pain and potentially death.

There is currently no cure or successful treatment for cancer metastases to bone.

Bisphosphonates are currently being used to treat osteoporosis and other bone disorders, however, their mode of action is poorly understood.

Bisphosphonates consist of two phosphate groups and are structurally analogous to pyrophosphate; therefore, bisphosphonates have the ability to inhibit enzymes that utilize nucleotide trisphosphate and potentially cause cell death.

The activity of bisphosphonates in inhibiting metabolic enzymes and causing cell death is considered to be therapeutic, however it is also likely that they have toxic effects that do not contribute to the therapeutic effects.

However, this has not been proven.

The mechanism of osteocalcin's action has been difficult to elucidate due to, in part, the fact that it has no known enzymatic activities.

Structural determination of small proteins is rather difficult because (i) heavy atom derivatives tend to destroy the crystal and (ii) another method that involves formation of seleno-methionine, which is commonly used, cannot be used because the host that makes seleno-protein (E. coli) can not make Gla.

As well, protein structural aspects, such as a long or flexible C-terminus or N-terminus, can increase the difficulty of crystallization.

The mammalian osteocalcins also have a longer N-terminus and are more difficult to crystallize.

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